Important Drug Research Articles

Isolation and Characterization of Novel Huperzine-Producing Endophytic Fungi from Lycopodiaceae Species.

Huperzine A (HupA) is an important drug for treating Alzheimer's disease (AD) and is primarily extracted from the Huperzia serrata (Lycopodiaceae). Failures in the chemical synthesis of Hup and in vitro culture have put H. serrata in danger of extinction, and there is a need for an extensive investigation of Hup from alternative perspectives. The aim of this study is to identify endophytic fungi that produce high Hup or simultaneously produce many types of Hup and have high genetic stability derived from other Lycopodiaceae species as a source of materials for natural Hup production. In this work, Hup-producing endophytic fungi were isolated from three species: Lycopodium clavatum, Phlegmariurus squarrosus, and P. phlegmaria. Of these, L. clavatum and P. squarrosus were confirmed as novel sources of Hup-producing fungi. Based on morphological characteristics and nuclear ribosomal DNA ITS sequences, four endophytic fungi Colletotrichum siamense THG1-17, Epicoccum sorghinum THG01-18, Phoma sp. TKH3-2, and Phyllosticta sp. THG2-27 were firstly isolated from these Lycopodiaceae plants, which were capable of simultaneously producing both HupA and HupB, as evidenced by high-performance liquid chromatography analysis. The four strains showed stability in Hup yield over 50 generations of culture with an in vitro storage period of 3 months. These isolated fungi will provide a new source of materials for further research to develop drugs containing HupA as well as HupB for AD treatment in the future.

Testosterone Biosynthesis from 4-Androstene-3,17-Dione Catalyzed via Bifunctional Ketoreductase

Testosterone (TS) is an important androgen drug and a precursor of steroid drug synthesis. Ketoreductase 2 (KR-2) (GenBank accession no. ABP64403.1) is observed to stereo-selectively catalyze the bioreduction of 4-androstene-3,17-dione (4-AD) to testosterone and contribute to the regeneration of NADH using isopropanol as a co-substrate. The Km value of KR-2 was 2.22 mmol/L with 4-AD, and the optimal pH was 6.5–7.0. The enzyme is stable and demonstrates relatively high-level enzyme activity at 40 °C. Acetone significantly inhibits this activity. This inhibition was overcome using an intermittent vacuum during the reaction process. Finally, the amount of TS reached 65.42 g/L after a 52 h reaction with 65.8 g/L 4-AD, 10% isopropanol, and 2 g/L β–NAD+ at 40 °C, with a conversion rate of 98.73%. A total of 6.15 g of TS was obtained from 6.58 g of 4-AD after the reaction and purification; the HPLC purity was 99.82%, and the overall yield was 92.81%. This enzyme provides a promising route for the green biosynthesis of testosterone for industrial applications.

Single-nucleotide polymorphism profiling by multimodal-targeted next-generation sequencing in methotrexate-resistant and -sensitive human osteosarcoma cell lines

Introduction: Methotrexate (MTX) is one of the most important drugs included in the first-line protocols to treat high-grade osteosarcoma (HGOS). Although several polymorphisms have been reported to be associated with drug response or MTX-related toxicity in pharmacogenetic studies, their role in the development of MTX resistance in HGOS is still unclear.Methods: Therefore, in this study, 22 single nucleotide polymorphisms (SNPs) in 4 genes of the folate metabolism, 7 MTX transporter genes, and 2 SNPs of the tumor protein p53 (TP53) gene were investigated using a custom multimodal-targeted next-generation sequencing (mmNGS) approach in 8 MTX-resistant and 12 MTX-sensitive human HGOS cell lines. The panel was validated by TaqMan genotyping assays.Results: High instability of TP53 rs1642785 was observed in all U-2OS/MTX variants. Allele changes of the solute carrier family 19 member 1/replication factor C subunit 1 (SLC19A1, previously known as RFC1) and rs1051266 were identified in all Saos-2/MTX-resistant variants in both DNA- and RNA- derived libraries compared to the parental Saos-2 cell line. Allele changes of methylenetetrahydrofolate reductase (MTHFR) rs1801133 were identified only in the RNA-derived libraries of the two U2OS variants with the highest MTX resistance level. Significantly upregulated gene expression associated with the development of MTX resistance was revealed for dihydrofolate reductase (DHFR) whereas SLC19A1 was downregulated. In addition, a fusion transcript of DHFR (ex4) and MutS Homolog 3 (MSH3) (ex9) was identified in the RNA libraries derived from the two U-2OS variants with the highest MTX resistance level.Conclusion: This innovative mmNGS approach enabled the simultaneous exploration of SNPs at DNA and RNA levels in human HGOS cell lines, providing evidence of the functional involvement of allele changes associated with the development of MTX resistance.

Associations between Selected ADRB1 and CYP2D6 Gene Polymorphisms in Children with Ventricular and Supraventricular Arrhythmias.

Background and Objectives: Tachycardia is a common cardiovascular disease. Drugs blocking β1-adrenergic receptors (ADRB1) are used in the therapy of arrhythmogenic heart diseases. Disease-related polymorphisms can be observed within the ADRB1 gene. The two most important are Ser49Gly and Arg389Gly, and they influence the treatment efficacy. The family of the cytochrome P450 system consists of the isoenzyme CYP2D6 (Debrisoquine 4-hydroxylase), which is involved in phase I metabolism of almost 25% of clinically important drugs, including antiarrhythmic drugs. A study was conducted to detect the ADRB1 and CYP2D6 gene polymorphisms. Materials and Methods: The material for the test was whole blood from 30 patients with ventricular and supraventricular tachycardia and 20 controls. The samples were obtained from the Department of Pediatric Cardiology. The first to be made was the extraction of DNA using a GeneMATRIX Quick Blood DNA Purification Kit from EURx. The selected ADRB1 and CYP2D6 gene polymorphisms were detected by high-resolution melting polymerase chain reaction (HRM-PCR) analysis. Results: Based on the analysis of melt profile data for each PCR product, the identification of polymorphisms was carried out. Heterozygotes and homozygotes were found in the examined alleles. Conclusions: The frequency of the Arg389Gly polymorphism differs statistically significantly between the control group and patients with supraventricular and ventricular arrhythmias, as well as between these two groups of patients. Moreover, the Arg389Gly polymorphism was statistically more prevalent in the group of girls with SVT arrhythmia compared to girls with VT. A few carriers of homozygous and heterozygous systems of the S49G polymorphism were detected among patients with arrhythmias, as well as control group. The percentage of individuals carrying the CYP2D6 4 allele as either homozygous or heterozygous was observed in the study and control groups. The high prevalence of the CYP2D6*4 allele carriers in both groups prompts the optimization of beta-1 blocker therapy.

Lobular Carcinoma of the Breast: A Comprehensive Review with Translational Insights.

The second most common breast carcinoma, invasive lobular carcinoma, accounts for approximately 15% of tumors of breast origin. Its incidence has increased in recent times due in part to hormone replacement therapy and improvement in diagnostic modalities. Although believed to arise from the same cell type as their ductal counterpart, invasive lobular carcinomas (ILCs) are a distinct entity with different regulating genetic pathways, characteristic histologies, and different biology. The features most unique to lobular carcinomas include loss of E-Cadherin leading to discohesion and formation of a characteristic single file pattern on histology. Because most of these tumors exhibit estrogen receptor positivity and Her2 neu negativity, endocrine therapy has predominated to treat these tumors. However novel treatments like CDK4/6 inhibitors have shown importance and antibody drug conjugates may be instrumental considering newer categories of Her 2 Low breast tumors. In this narrative review, we explore multiple pathological aspects and translational features of this unique entity. In addition, due to advancement in technologies like spatial transcriptomics and other hi-plex technologies, we have tried to enlist upon the characteristics of the tumor microenvironment and the latest associated findings to better understand the new prospective therapeutic options in the current era of personalized treatment.

Serendipitous discovery of Class I HDAC inhibitors from rational design of molecular glue degraders targeting HDAC

Zinc-dependent histone deacetylases (HDACs) play an essential role as epigenetic regulators and are becoming increasingly important drug targets for the treatment of cancer. Although five HDAC inhibitors have been approved for treating several cancers, only one of them is a Class I HDAC inhibitor, which may have advantages over pan-HDAC inhibitors due to the various side effects associated with the latter. On the other hand, the emerging strategy of molecular glue degraders offers a unique advantage for targeting therapeutic proteins. In this study, we synthesized a series of candidate compounds based on the molecule glue, pomalidomide, using a “merger principle”, initially aiming to obtain molecular glue degraders that can target HDAC degradation. However, we serendipitously discovered that compounds 2f and 3f may be potent Class I HDAC selective inhibitors. After further evaluation, we found that compounds 2f and 3f exhibit selective inhibitory effects on Class I HDAC in cancer cells. Moreover, they showed the robust antiproliferative activities against various hematological tumor cells, comparable to that of the approved Class I HDAC inhibitor, Chidamide. These results suggest that pomalidomide-derivatized compounds have promising potential as Class I HDAC inhibitors with therapeutic applications in cancer treatment.

Dual-function transaminases with hybrid nanoflower for the production of value-added chemicals from biobased levulinic acid.

The U.S. Department of Energy has listed levulinic acid (LA) as one of the top 12 compounds derived from biomass. LA has gained much attention owing to its conversion into enantiopure 4-aminopentanoic acid through an amination reaction. Herein, we developed a coupled-enzyme recyclable cascade employing two transaminases (TAs) for the synthesis of (S)-4-aminopentanoic acid. TAs were first utilized to convert LA into (S)-4-aminopentanoic acid using (S)-α-Methylbenzylamine [(S)-α-MBA] as an amino donor. The deaminated (S)-α-MBA i.e., acetophenone was recycled back using a second TAs while using isopropyl amine (IPA) amino donor to generate easily removable acetone. Enzymatic reactions were carried out using different systems, with conversions ranging from 30% to 80%. Furthermore, the hybrid nanoflowers (HNF) of the fusion protein were constructed which afforded complete biocatalytic conversion of LA to the desired (S)-4-aminopentanoic acid. The created HNF demonstrated storage stability for over a month and can be reused for up to 7 sequential cycles. A preparative scale reaction (100mL) achieved the complete conversion with an isolated yield of 62%. Furthermore, the applicability of this recycling system was tested with different β-keto ester substrates, wherein 18%-48% of corresponding β-amino acids were synthesized. Finally, this recycling system was applied for the biosynthesis of pharmaceutical important drug sitagliptin intermediate ((R)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid) with an excellent conversion 82%.

Molecular identification of multiple drug resistance (MDR) strain of Mycobacterium tuberculosis.

Isoniazid and rifampin are the first -line drugs against Mycobacterium tuberculosis. Resistance to these important drugs is a serious threat to human public health. Therefore, this study aimed at molecular detection of resistance to these valuable drugs. In this descriptive cross-sectional study, 111 non - duplicated clinical samples including sputum and Bronchoalveolar lavage (BAL) samples were collected from patients referred to the Ardabil Health Center between 2017 and 2020. The samples were first examined by microscopic method, then their DNA was extracted using the boiling method. Specific primers and MAS-PCR method were employed for the detection resistance to isoniazid and rifampin drugs and identification of MDR strain. of 111 specimens, 15.3% belonged to NTM. In total, the resistance rate to isoniazid and rifampin was 17% and 27% respectively while the resistance rate to isoniazid and rifampin among NTM was 61.54% and 38.46%. In our study, the prevalence of resistance to isoniazid and rifampin among Mycobacterium tuberculosis complex(MTC) and non-tuberculous mycobacteria(NTM) was investigated using the MAS-PCR method. This work highlighted the high anti- tuberculosis resistance rate among NTM compared to MTC strains.

Fungal diversity and drug susceptibility of the oral mycobiome of domestic dogs.

The purpose of this study was to characterize the variety and diversity of the oral mycobiome of domestic dogs and to identify the commensal and potentially pathogenic fungi present. Two hundred fifty-one buccal swabs from domestic dogs were obtained and struck onto a chromogenic fungal growth medium that distinguishes between fungal species based on colony color and morphology. After isolating and harvesting single colonies, genomic DNA was extracted from pure cultures. PCR was used to amplify a fungal-specific variable rDNA region of the genome, which was then sent for sequencing. Sequencing results were input into the NCBI BLAST database to identify individual components of the oral mycobiome of tested dogs. Of the 251 dogs swabbed, 73 had cultivable fungi present and 10 dogs had multiple fungal species isolated. Although the dogs did not show signs of oral infections at the time, we did find fungal species that cause pathogenicity in animals and humans. Among fungal isolates, Malassezia pachydermatis and species from the genus Candida were predominant. Following fungal isolate identification, antifungal drug susceptibility tests were performed on each isolate toward the medically important antifungal drugs including fluconazole, ketoconazole, and terbinafine. Drug susceptibility test results indicated that a large number of isolates had high MIC values for all three drugs. Exploring the oral mycobiome of dogs, as well as the corresponding drug susceptibility profiles, can have important implications for canine dental hygiene, health, and medical treatment. Identifying the microorganisms within the canine mouth can illustrate a common pathway for fungal pathogens of One Health concern to spread from our canine companions to humans.

Smart Magnetic Nanocarriers for Codelivery of Nitric Oxide and Doxorubicin for Enhanced Apoptosis in Cancer Cells.

Extremely short half-life therapeutic molecule nitric oxide (NO) plays significant roles in the functioning of various physiological and pathological processes in the human body, whereas doxorubicin hydrochloride (DOX) is a clinically important anticancer drug widely used in cancer chemotherapy. Thus, the intracellular delivery of these therapeutic molecules is tremendously important to achieve their full potential. Herein, we report a novel approach for the development of highly water-dispersible magnetic nanocarriers for codelivery of NO and DOX. Primarily, bifunctional magnetic nanoparticles enriched with carboxyl and thiol groups were prepared by introducing cysteine onto the surface of citrate-functionalized Fe3O4 nanoparticles. DOX was electrostatically conjugated onto the surface of bifunctional nanoparticles via carboxyl moieties, whereas the thiol group was further nitrosated to provide NO-releasing molecules. The developed magnetic nanocarrier exhibited good aqueous colloidal stability, protein resistance behavior, and high encapsulation efficacy for NO (65.5%) and DOX (85%), as well as sustained release characteristics. Moreover, they showed superior cytotoxicity toward cancer (A549 and MCF-7) cells via apoptosis induction over normal (WI26VA4) cells. Specifically, we have developed magnetic nanocarriers having the capability of dual delivery of NO and DOX, which holds great potential for combinatorial cancer treatment.

Identification of a carbohydrate recognition motif of purinergic receptors.

As a major class of biomolecules, carbohydrates play indispensable roles in various biological processes. However, it remains largely unknown how carbohydrates directly modulate important drug targets, such as G-protein coupled receptors (GPCRs). Here, we employed P2Y purinoceptor 14 (P2Y14), a drug target for inflammation and immune responses, to uncover the sugar nucleotide activation of GPCRs. Integrating molecular dynamics simulation with functional study, we identified the uridine diphosphate (UDP)-sugar-binding site on P2Y14, and revealed that a UDP-glucose might activate the receptor by bridging the transmembrane (TM) helices 2 and 7. Between TM2 and TM7 of P2Y14, a conserved salt bridging chain (K2.60-D2.64-K7.35-E7.36 [KDKE]) was identified to distinguish different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine. We identified the KDKE chain as a conserved functional motif of sugar binding for both P2Y14 and P2Y purinoceptor 12 (P2Y12), and then designed three sugar nucleotides as agonists of P2Y12. These results not only expand our understanding for activation of purinergic receptors but also provide insights for the carbohydrate drug development for GPCRs.

Development of a new class of potent and highly selective G protein-coupled receptor kinase 5 inhibitors and structural insight from crystal structures of inhibitor complexes

G protein-coupled receptor kinase 5 (GRK5) is an important drug development target for heart failure, cardiac hypertrophy, and cancer. We have designed and developed a new class of highly selective, potent, and non-covalent GRK5 inhibitors. One of the inhibitors displayed GRK5 IC50 value of 10 nM and exhibited >100,000-fold selectivity over GRK2. The X-ray structure of a ketoamide-derived inhibitor-bound GRK5 showed the formation of a hemithioketal intermediate with active site Cys474 in the GRK5 active site and provided new insights into the ligand-binding site interactions responsible for high selectivity. The current studies serve as an important guide to therapeutic GRK5 inhibitor drug development.

SGLT2 inhibitors and vericiguat in real-world patients admitted for worsening heart failure

Abstract Introduction Worsening heart failure (WHF) is an emerging concept that is defined by escalating signs and symptoms of heart failure (HF) in patients with chronic heart failure (CHF) despite stable therapy. After a WHF event, there is a vulnerable period in which the risk of hospitalization and mortality is high. Sodium-glucose-co-transporter inhibitors (SGLT2i) and vericiguat, a soluble guanylate cyclase stimulator (sGCs), have been investigated in patients with WHF bother during hospitalization (EMPULSE) and right after the discharge (VICTORIA and SOLOIST-WHF) in the management of this condition with positive results. Aims This study aims to investigate the eligibility of VICTORIA, SOLOIST-WHF and EMPULSE in real-world population of patients admitted for worsening heart failure (WHF) and to analyze the differences between these groups of patients. Material and methods In this retrospective cohort study, 275 patients admitted for worsening heart failure in our cardiology department between 2017 and 2022 were enrolled. The percentages of eligible patients according to VICTORIA, SOLOIST-WHF and EMPULSE inclusion and exclusion criteria were analyzed, then the differences between the characteristics of our population and trials population were evaluated. Results 83 patients (30,2%) were eligible according to VICTORIA trial criteria, 35 patients (12,7%) according to SOLOIST-WHF and 186 patients (67,6%) according to EMPULSE. Limiting factors were ejection fraction (EF) > 45% in VICTORIA, the presence of diabetes in SOLOIST-WHF and the cut-off levels of NT-proBNP and BNP. Our patients were older (mean 78 years and median 80 years) than trials populations (67 years; 69 years; 71 years). EF was higher (mean 38% and median 35%) than VICTORIA (29%) and than EMPULSE (31%), but similar compared to SOLOIST-WHF (35%). Estimated glomerular filtration rate (eGFR) was lower compared to VICTORIA (56,0 ml/min vs 61,5 ml/min) whereas higher compared to EMPULSE and SOLOIST (54,0 ml/min vs 49,2 ml/min vs 50,0 ml/min). Diabetes was less common in our population (32,7% vs 47,1% vs 100% vs 46,8%); on the other hand, there were higher levels of NT-proBNP (median 5080 pg/ml) in our population compared to trials (3377 pg/ml; 1816,8 pg/ml; 3299 pg/ml). Conclusion SGLT2i and vericiguat are important drugs in the management of WHF. At the moment, vericiguat can be used only in patients with EF < 45% and this is the most limiting factor, whereas SGLT2i can be used regardless of EF. These data show that their applicability is high in patients with WHF and that the characteristics of trials populations reflect a real-world population. More data are needed about the use of vericiguat and SGLT2i, mostly in the first days of hospitalization. Recommendations for how to manage patients with WHF must be defined, national and international registries of HF hospitalizations may resolve this issue.Eligibility of vericiguat and SGLT2iComparison of baseline characteristics

Left ventricular reverse remodeling following initiation of sacubitril/valsartan for heart failure with reduced ejection fraction and low blood pressure

Abstract Background Sacubitril/valsartan has become an important first-line drug for symptomatic heart failure (HF) patients, especially with left ventricular (LV) ejection fraction (LVEF) < 50%. Since sacubitril/valsartan was reported to be more potent than renin-angiotensin-aldosterone system inhibitors for lowering blood pressure, HF patients with low blood pressure were not included in most clinical studies as a result of sacubitril/valsartan administration, and average systolic blood pressure of most enrolled patients was around 120 mmHg. Thus, the effect of sacubitril/valsartan on cardiovascular outcomes, especially LV reverse remodeling for HF patients with LVEF < 50% and low blood pressure, remains uncertain. Purpose The aim of this study was to examine the effect of sacubitril/valsartan on LV reverse remodeling after initiation of the maximum tolerated dose of sacubitril/valsartan for HF patients with LVEF < 50% and systolic blood pressure ≤ 100 mmHg, and a comparison of its efficacy with that for patients with systolic blood pressure > 100 mmHg. Methods We retrospectively studied 164 HF patients with LVEF < 50% who were treated with sacubitril/valsartan from two institutions. Echocardiography was performed before and 9.5 ± 5.1 months after initiation of maximum tolerated dose of sacubitril/valsartan. Results sacubitril/valsartan was discontinued for 17 (10.4%) as decided by the attending physician mainly due to hypotension. The prevalence of discontinuation of sacubitril/valsartan for the low blood pressure group was higher than that for the non-low blood pressure group, but the difference was not statistically significant (16.0% vs. 9.4%, P = 0.32). No serious adverse events occurred during this study. The maximum tolerated dose of sacubitril/valsartan was lower for the low blood pressure group than for the non-low blood pressure group (165 ± 106 mg vs. 238 ± 124 mg, P = 0.017). As expected, significant LV reverse remodeling was observed in the non-low blood pressure group after initiation of sacubitril/valsartan. It was noteworthy that significant LV reverse remodeling was also observed in the low blood pressure group after initiation of sacubitril/valsartan (LV end-diastolic volume: 177.3 ± 66.0 mL vs. 137.7 ± 56.1 mL, P<0.001, LV end-systolic volume: 131.6 ± 60.3 mL vs. 94.6 ± 55.7 mL, P<0.001, LVEF: 26.8 ± 10.3% vs. 33.8 ± 13.6%, P = 0.015). Relative changes in LV volumes and LVEF after initiation of sacubitril/valsartan were similar for the two groups (Figure). Conclusions Significant LV reverse remodeling can occur after initiation of sacubitril/valsartan even in HF patients with LVEF < 50% and systolic blood pressure ≤ 100 mmHg if tolerated. This finding is expected to offer new insights for better management of HF patients.Figure

Is the Functional Response of a Receptor Determined by the Thermodynamics of Ligand Binding?

For an effective drug, strong binding to the target protein is a prerequisite, but it is not enough. To produce a particular functional response, drugs need to either block the proteins' functions or modulate their activities by changing their conformational equilibrium. The binding free energy of a compound to its target is routinely calculated, but the timescales for the protein conformational changes are prohibitively long to be efficiently modeled via physics-based simulations. Thermodynamic principles suggest that the binding free energies of the ligands with different receptor conformations may infer their efficacy. However, this hypothesis has not been thoroughly validated. We present an actionable protocol and a comprehensive study to show that binding thermodynamics provides a strong predictor of the efficacy of a ligand. We apply the absolute binding free energy perturbation method to ligands bound to active and inactive states of eight G protein-coupled receptors and a nuclear receptor and then compare the resulting binding free energies. We find that carefully designed restraints are often necessary to efficiently model the corresponding conformational ensembles for each state. Our method achieves unprecedented performance in classifying ligands as agonists or antagonists across the various investigated receptors, all of which are important drug targets.

MicroED as a Powerful Tool for Structure Determination of Macrocyclic Drug Compounds Directly from Their Powder Formulations.

Macrocycles are important drug leads with many advantages including the ability to target flat and featureless binding sites as well as to act as molecular chameleons and thereby reach intracellular targets. However, due to their complex structures and inherent flexibility, macrocycles are difficult to study structurally, and there are limited structural data available. Herein, we use the cryo-EM method MicroED to determine the novel atomic structures of several macrocycles that have previously resisted structural determination. We show that structures of similar complexity can now be obtained rapidly from nanograms of material and that different conformations of flexible compounds can be derived from the same experiment. These results will have an impact on contemporary drug discovery as well as natural product exploration.

A transcriptomics-based drug repositioning approach to identify drugs with similar activities for the treatment of muscle pathologies in spinal muscular atrophy (SMA) models.

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by the reduction of survival of motor neuron (SMN) protein levels. Although three SMN-augmentation therapies are clinically approved that significantly slow down disease progression, they are unfortunately not cures. Thus, complementary SMN-independent therapies that can target key SMA pathologies and that can support the clinically approved SMN-dependent drugs are the forefront of therapeutic development. We have previously demonstrated that prednisolone, a synthetic glucocorticoid (GC) improved muscle health and survival in severe Smn-/-;SMN2 and intermediate Smn2B/- SMA mice. However, long-term administration of prednisolone can promote myopathy. We thus wanted to identify genes and pathways targeted by prednisolone in skeletal muscle to discover clinically approved drugs that are predicted to emulate prednisolone's activities. Using an RNA-sequencing, bioinformatics, and drug repositioning pipeline on skeletal muscle from symptomatic prednisolone-treated and untreated Smn-/-; SMN2 SMA and Smn+/-; SMN2 healthy mice, we identified molecular targets linked to prednisolone's ameliorative effects and a list of 580 drug candidates with similar predicted activities. Two of these candidates, metformin and oxandrolone, were further investigated in SMA cellular and animal models, which highlighted that these compounds do not have the same ameliorative effects on SMA phenotypes as prednisolone; however, a number of other important drug targets remain. Overall, our work further supports the usefulness of prednisolone's potential as a second-generation therapy for SMA, identifies a list of potential SMA drug treatments and highlights improvements for future transcriptomic-based drug repositioning studies in SMA.

Integrin αvβ8-Mediated Activation of TGF-β1 Is Involved in Regulating the Sustained Response after TPO-Ras Withdrawal in ITP Patients

Background: Thrombopoietin receptor agonists（TPO-RAs）are effective and safe in immune thrombocytopenia (ITP), and are currently the most important second-line drugs. The effect of TPO-RAs is transient, platelet counts usually return to baseline within 2 weeks unless treatment is continued. Only one-third of patients can maintain after drug discontinuation, but the mechanism is unknown and worthy of further exploration. Method and Results：In this study we observed 66 patients treated with TPO-RAs and followed-up to three months after drug withdrawal. Patients were divided into three groups: non-response group (NR), sustained group (Group A), and non- sustained group (Group B) (Fig1-A). The blood samples were collected at the drug discontinuation, and we found that compared with total TGF-β1 levels，plasma activated TGF-β1 levels were extremely lower in NR patients and were also reduced in group B (Fig1-B,C). At the same time, regulatory T cells have the same trend. By using the qPCR and Western blot we preliminary found that integrin αvβ8 regulate the activation of TGF-β1 to promote the formation of Tregs, which affects the sustained response to TPO-RAs withdrawal. Furthermore, lower levels of activated TGF-β1 in the bone marrow were associated with higher numbers of megakaryocytes, fewer plate-producing megakaryocytes, and decreased proportion of hyperploid megakaryocytes (≥16N). Through in vitro cell culture, we found that recombinant human integrin avb8 protein can promote Treg differentiation and inhibit T cell activation in ITP patients' PBMCs, but this phenomenon can be reversed by anti-β8 antibody. Then, we constructed PF4-TGF-β1 conditional knockout mice (Cre+ mice) and constructed of an ITP passive model by injecting CD41 antibody and found that the Cre+ mice have higher platelets at baseline, while the platelets dropped rapidly than Cre- mice after CD41 antibody injection (Fig2-A). Using rhTPO to treat the ITP mice, we found that Cre+ mice were less effective than Cre- mice, and the sustained response time was shorter after drug withdrawal. When sacrificed the mice, decreased Treg cells and elevated M1 macrophages were detected in Cre+ mice. Meanwhile, HE staining of bone marrow found a greater number of megakaryocytes, but the cell diameter decreased significantly (Fig2-B, D). Next, we treated the mice with rhTPO in combined with recombinant mice integrin avb8 (50ng/kg or 250ng/kg), the therapeutic efficacy and sustained response time in the high-dose avb8 group were significantly improved (Fig2-C) We also constructed a more representative ITP active model of autoimmune diseases, by immunizing CD61KO mice to produce autoantibodies and infusing spleen cells back into SCID mice. Simultaneously through transfection siRNA or shRNA to activate or silence mouse T cell integrin β8 and reinfuse it into SCID mice. We found that activating integrin β8 in mice had a more significant therapeutic effect and maintained a longer response time. Finally, through single-cell sequencing analysis, the smads signaling pathways，integrin binding pathways and AP-1 family signaling pathways were significantly enriched in group A patients. Further study confirmed that TPO-RAs can regulate the AP-1 family, which upregulated the β8 subunit, resulting in enhanced activation of TGF-β1 (Fig3-A, B). Conclusion: Integrin αvβ8 activates TGF-β1 and upregulates Tregs to restore immune intolerance in ITP patients, meanwhile, activated TGF-β1 also promotes hyperploidy megakaryocyte differentiation in bone marrow, which co-regulates the sustained response after TPO-RAs discontinuation.

Correlation between Disease Biomarkers and Hemoglobin F Levels in Sickle Cell Patients

Introduction Sickle cell disease (SCD) is a hemoglobinopathy that impacts 100,000 people in the United States (Kavanagh, 2022). SCD is associated with various complications, some of which include stroke, venous thrombosis, cholelithiasis, and liver damage. Different biomarkers can be measured that can be useful in assessing the presence and progression of these complications including T.bili, LDH, AST, ALT, and albumin. Hydroxyurea is a tremendously important drug in the management of patients with sickle cell complications due to its ability to stimulate production of fetal hemoglobin (HbF). It has been proven to reduce the frequency of sickle cell crisis (Osunkwo, 2020). Limited research exists that correlates hemoglobin F values to these biomarkers. The aim of this study was to determine if there is a correlation between HbF levels and these biomarkers in patients with SCD. Methods A database of 496 SCD patients at the Center for Blood Disorders in Augusta, Georgia was analyzed in this cross-sectional study. Among the information extracted were data surrounding common clinical complications of SCD (i.e.thrombotic events, pulmonary hypertension, avascular necrosis, etc), anticoagulant use, disease biomarkers (i.e. D-dimer, T. bili, LDH, ALT/AST), history of COVID-19, and percentage of HbF and HbS. In patients with no history of thrombotic event, the most recent data was extracted. Pearson's correlation coefficient and Fisher's z-test were used to test the association between HbF and LDH, T.bili, AST, ALT, and albumin levels using R version 4.2.3 Results 483 of the 496 patients had a recorded T.bili level. The mean HbF for patients with a normal T.bili was 8.51 (SD 9.58). The mean HbF for patients with an elevated T.bili was 9.55 (SD 9.59). A two-sample t-test comparing the two yielded a p-value of 0.4066. 200 of the 496 patients had a recorded LDH. The mean HbF for patients with a normal LDH was 7.65 (SD 7.64). The mean HbF for patients with an elevated LDH was 9.74 (SD 8.32). A two-sample t-test comparing the two yielded a p-value of 0.1306. 482 of the 496 patients had a recorded AST level. The mean HbF for patients with a normal AST was 9.70 (SD 10.89). The mean HbF for patients with an elevated AST was 8.69 (SD 7.35). 483 of the 496 patients had a recorded ALT level. The mean HbF for patients with a normal ALT was 9.11 (SD 9.72). The mean HbF for patients with an elevated ALT was 10.34 (SD 9.77). 127 of the 496 patients had a recorded albumin level. The average HbF for patients with a normal albumin was 9.30 (SD 9.66). The average HbF for patients with a low albumin was 8.70 (SD 8.92). Conclusion Laboratory biomarkers such as LDH, T.bili, AST, ALT, and albumin are used to monitor various complications associated with SCD such as cholelithiasis and liver damage, respectively. Although there have been prior studies that demonstrate higher levels of HbF are correlated with better patient outcomes, we observed that there is no significant relationship between HbF and biomarker levels of T.bili, LDH, AST, ALT and albumin in these 496 SCD patients. These findings suggest that there may be factors other than HbF levels that play a role in the presence and progression of complications in patient with SCD.

Clinical Similarity of Biosimilars and Reference Drugs: A Comprehensive Review and New Hope for Public Health in a New Frontier.

Patents and exclusive rights on reference biologics contribute to the emergence of biosimilars. Regulatory bodies, such as the Food and Drug Administration (FDA), World Health Organization (WHO), and EMA (European Medicines Agency) for assessing clinical safety, effectiveness, and consequences between biosimilars and reference medications, have established guidelines. Since generic small molecules from reference can be easily swapped, biosimilars cannot be used interchangeably and may not always indicate interchangeability due to highly restrictive properties. It can be replaced with a reference without the healthcare provider's help under the interchangeability context. The purpose of our study is to analyze and compare evidence-based clinical safety, therapeutic potential, and importance (outcomes) of several biosimilars with their references along with clinical uses in chronic diseases. Through a comprehensive systemic literature review of more than 100 articles involving medicinally important drugs whose bio-similarity works optimally, safety-efficacy parameters have been analyzed. Analysis of biosimilar usage, approval, and safety-efficacy aspects are majorly focused upon herein in this review. From this systemic review, it can be stated that the majority of biosimilars are clinically and statistically equivalent to their originators. As biosimilars have good safety-efficacy aspects with lower prices, their utilization can be more encouraged, which was already done by the FDA with the establishment of a public online database entitled "Purple Book," which includes all information regarding biological drugs. To conclude, we suggest widespread use of high-grade biosimilars in clinical practice, maybe via changing, exchanging, or switching, with appropriate clinical monitoring and pharmacovigilance to improve patient accessibility to modern medicines, as it provides similar efficacy and safety parameters across all the accumulated clinical trials and studies.